CN116716251A - A single cell processing method for vitreous samples and its application - Google Patents

Abstract

The invention provides a single-cell processing method of a vitreous sample and application thereof. The single-cell processing method of the vitreous body sample comprises the following steps of S1, digestive juice composition of the vitreous body sample: comprises one or more of collagenase, pancreatin and dispase, wherein the concentration is 0.1% -2%, and 0.5% -1.5% FBS is dissolved in PBS; s2, immediately placing the patient vitreous body sample on ice for transportation, transferring the tissue into a 50mL centrifuge tube, and adding 10ml of digestive juice in the S1; s3, placing the centrifuge tube in a shaking table for digestion; s4, after digestion, adding 0.5-1.5 ml of FBS and 2.5-10 ml of PBS to terminate; s5, then centrifuging at 800g for 5min, and carefully discarding the supernatant; s6, diluting the split red with 5-time volume PBS for 1-5 min, and centrifuging. The single-cell processing method and the application of the vitreous body sample provided by the invention have the advantages of capability of obtaining active cells with high activity and high purity, simplicity and rapidness in operation, no need of expensive instruments and reagents and high cell activity rate.

Description

A single cell processing method for vitreous samples and its application

Technical field

The present invention relates to the technical field of single cell processing, and in particular to a single cell processing method for vitreous samples and its application.

Background technique

Primary vitreoretinal lymphoma (PVRL) is a subtype of primary central nervous system lymphoma (primary central nervous system lymphoma (PCNSL)). It is a rare and fatal ocular malignant tumor. The pathological type of 97% of patients is Aggressive B-cell lymphoma. PVRL is often misdiagnosed as uveitis, with 94.5% of the affected eyes showing white punctate vitreous opacity, and 60.3% of the affected eyes showing yellow-white subretinal infiltrates. Due to the difficulty of intraocular specimen collection and pathological diagnosis, the average diagnosis time of this disease is 13.4 months.

The gold standard for PVRL diagnosis is pathological diagnosis. However, PVRL is a non-solid tumor with a large number of lymphoma cells floating in the vitreous cavity and scattered under the retina. To confirm the diagnosis, vitrectomy is required, and about 1.5ml of vitreous fluid is removed. The vitreous body is a colorless and transparent colloid, mainly composed of water, hyaluronic acid and collagen fibers. Lymphoma cells adhere to the vitreous collagen fibers and are difficult to dissociate into single cells. Moreover, the tumor cells are extremely fragile and cannot be separated from the body. It thaws within 2 hours and loses its original cell shape. Therefore, the positive rate of pathological diagnosis is relatively low, and is replaced by different molecular or genetic diagnosis.

In order to improve the positive rate of pathological diagnosis and study the biological behavior of tumor cells, an effective method is urgently needed to separate lymphoma cells from the colloid vitreous, and the purity, quantity and activity of the cells need to be ensured. Therefore, it is necessary to provide A new single-cell processing method for vitreous samples and its application solve the above technical problems.

Contents of the invention

The technical problem solved by the present invention is to provide a single cell processing method and application for vitreous samples that can obtain high activity and high purity active cells, is simple and fast to operate, does not require expensive equipment and reagents, and has high cell viability. .

In order to solve the above technical problems, the single cell processing method of vitreous samples and its application provided by the present invention include: S1. The digestive juice of the vitreous sample is composed of: one or a combination of two or more types of collagenase, trypsin and dispase. , with a concentration of 0.1%-2%, and 0.5%-1.5% FBS dissolved in PBS; S2. Collect the patient's vitreous sample and immediately place it on ice for transportation, transfer the tissue into a 50mL centrifuge tube, and add 10ml of the digestive fluid in S1; S3. Place the centrifuge tube on a shaker for digestion; S4. After digestion, add 0.5-1.5ml FBS and 2.5-10ml PBS to terminate; S5. Then centrifuge at 800gx5min and carefully discard the supernatant; S6. After 1-5min of split red, use 5 Dilute with twice the volume of PBS and centrifuge; S7, wash once with 0.02% ~ 0.06% PBS-BSA or PBS containing 0.5% ~ 1.5% FBS, centrifuge at 500gx5min; S8, after washing, resuspend with 0.04% PBS-BSA, set aside After staining with pan blue and counting.

Preferably, the digestion temperature of the shaking table in S3 is 37°C, the rotation speed is 50-100 rpm, and the digestion time is 10-60 minutes.

Preferably, the volume of the patient's vitreous sample collected in S2 is 1 ml.

Compared with related technologies, the single cell processing method for vitreous samples provided by the present invention and its application have the following beneficial effects:

The invention provides a single cell processing method for vitreous samples and its application, which can obtain high-activity, high-purity active cells. The operation is simple and fast, and does not require expensive equipment and reagents. The cell viability rate can reach up to 84%. The separation and digestion method of the present invention is efficient and fast, and has successfully extracted highly active and pure lymphoma cells from the vitreous body for single-cell sequencing research, and is suitable for popularization and application.

Description of the drawings

Figure 1 is a diagram of active cells obtained by the single cell processing method of vitreous samples provided by the present invention and its application.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

Please refer to Figure 1 in conjunction with the single cell processing method and application of vitreous samples including: S1. The composition of the digestive fluid of vitreous samples is: including one or two or more combinations of collagenase, trypsin and dispase, with a concentration of 0.1 %-2%, and 0.5% to 1.5% FBS dissolved in PBS; S2. Collect the patient's vitreous samples and immediately place them on ice for transportation. Taking vitreous lymphoma samples as an example, digest and process to obtain a single cell suspension for single cell For transcriptome sequencing research, transfer the tissue into a 50mL centrifuge tube and add 10ml of the digestion solution in S1; S3, place the centrifuge tube on a shaker for digestion; S4, after digestion, add 1mlFBS and 5mlPBS to terminate; S5, then centrifuge at 800gx5min. Carefully discard the supernatant; S6, after 1 to 5 minutes of splitting the red, dilute with 5 times the volume of PBS and centrifuge; S7, wash once with 0.02% to 0.06% PBS-BSA or PBS containing 0.5% to 1.5% FBS, and centrifuge at 500gx5min ; S8. After washing, resuspend with 100 μL 0.04% PBS-BSA. After trypan blue staining and counting, the bright spots in the picture are active cells.

The digestion temperature of the shaking table in S3 is 37°C, the rotation speed is 50-100 rpm, and the digestion time is 10-60 minutes.

The volume of the patient's vitreous sample collected in S2 was 1 ml.

Single-cell processing of vitreous samples for applications such as single-cell sequencing or flow cytometry.

Compared with related technologies, the single cell processing method for vitreous samples provided by the present invention and its application have the following beneficial effects:

The invention provides a single cell processing method for vitreous samples and its application, which can obtain high-activity, high-purity active cells. The operation is simple and fast, and does not require expensive equipment and reagents. The cell viability rate can reach up to 84%. The separation and digestion method of the present invention is efficient and fast, and has successfully extracted highly active and pure lymphoma cells from the vitreous body for single-cell sequencing research, and is suitable for popularization and application.

The above are only examples of the present invention, and do not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the description and drawings of the present invention, or directly or indirectly applied to other related technologies fields are equally included in the scope of patent protection of the present invention.

Claims (4)

1. A method for single cell processing of a vitreous sample, comprising the steps of:

s1, the digestive juice composition of the vitreous body sample is as follows: comprises one or more of collagenase, pancreatin and dispase, wherein the concentration is 0.1% -2%, and 0.5% -1.5% FBS is dissolved in PBS;

s2, immediately placing the patient vitreous body sample on ice for transportation, transferring the tissue into a 50mL centrifuge tube, and adding 10ml of digestive juice in the S1;

s3, placing the centrifuge tube in a shaking table for digestion;

s4, after digestion, adding 0.5-1.5 ml of FBS and 2.5-10 ml of PBS to terminate;

s5, centrifuging at 800g×5min, and carefully discarding the supernatant;

s6, diluting with PBS (phosphate buffer solution) with 5 times of volume and centrifuging after 1-5 minutes of red burst;

s7, washing with 0.02% -0.06% PBS-BSA or PBS containing 0.5% -1.5% FBS for 1 time, and centrifuging 500g x5min;

s8, after washing, 100 mu L of 0.04% PBS-BSA was resuspended, and trypan blue was stained and counted.

2. The method for single-cell processing of a vitreous sample according to claim 1, wherein the shaking table digestion temperature in S3 is 37 ℃, the rotation speed is 50-100rpm, and the digestion time is 10-60min.

3. The method for single-cell processing of a vitreous sample and its use according to claim 2, wherein the volume of the vitreous sample of the patient collected in S2 is 1ml.

4. Use of the method of any one of claims 1-3 in single cell sequencing or flow cytometry detection, among others.